The goal of this project is to develop the next generation of targeted T-cells with chimeric antigen receptors (CARs) for use in carcinomas and hematologic malignancies. Therefore, these studies address the cancer epidemic that afflicts the population in the United States. CAR T cells are now beginning to show activity in a number of pilot clinical trials and they have significant potential for therapy of many cancers that are currently incurable. However two issues have emerged that provide a barrier to further rapid progress in the field: 1) the persistence of CAR T cells in patients with solid cancer has been limited, unlike the case with CARs that target CD19; 2) T cells become exhausted, become anergic or die in the toxic tumor microenvironment, unlike the case of hematologic malignancies, where CAR T cells have continued to function for at least 5 years in responding patients. Our preliminary data indicates that the metabolic profiles of CAR T cells can be altered at will by changing the design of the signaling domain in the CAR construct. In this project, we will use the principles of synthetic biology and the tools of lentiviral vector technology, mRNA electroporation technology, and clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated protein 9 (Cas9) technology to adapt the metabolism of T cells in order to promote survival in harsh tumor microenvironments. The theme of the project is that T cells with more potent and sustained antitumor effects can be designed to resist metabolic checkpoints such as hypoxia, hyperkalemia, acidosis, and glucose deprivation. In summary, these overlapping studies will test the central hypothesis that synthetically enhanced CAR T cells will improve CAR immunotherapy compared to therapy with currently available CAR T cells. At the conclusion of this project, a next generation of metabolically enhanced CAR T cells will be available for testing in pilot clinical trials in patients with advanced pancreatic cancer.